A DNA REPAIR/PHASE VARIATION REPORTER SYSTEM USING A POLY-GUANINE TRACT IN A NEISSERIA GONORRHOEAE NITROREDUCTASE GENE
Rodgers, Mark Andrew
Stein, Daniel C
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Neisseria gonorrhoeae undergoes phase variation to adapt to new environments, increase pathogenesis, and evade the host immune system. This may be due to defects in DNA repair. A reporter system was created to detect phase variation by phenotypic switching from a nitrofuran-sensitive phenotype to a nitrofuran-resistant (NitR) phenotype. Strains were created with poly-guanine tracts from 5 to 12 guanines in the coding region of a nitroreductase gene (nfsB) that would be susceptible to frame-shifting mutations during DNA replication. The minimum number of consecutive guanines needed to observe increased mutation was 5. A strain expressing 7 guanines nfsB possessed nitroreductase activity similar to wild-type and a spontaneous mutation frequency that was increased ~104 fold relative to wild-type. Frame-shifting mutations of strain expressing 8 guanines in nfsB were observed using denaturing gradient gel electrophoresis (DGGE). Future work with the reporter system could lead to new understanding of phase variation and DNA repair.